Protein C is a vitamin K-dependent protein that is synthesized in the liver and circulates as an inactive zymogen in a concentration of 4 mg/l. It is transformed by the thrombin-thrombomodulin complex into the active serine protease (activated Protein C) on the vessel wall surface (endothelium). It is known that activated Protein C has profibrinolytic properties. It also has anticoagulatory effects because it inactivates Factor Va, the co-factor for the Factor Xa-induced prothrombin activation (thrombin formation), and Factor VIIIa, the co-factor for Factor IXa-induced Factor X activation, by proteolysis.
The activation of Protein C in vivo constitutes a negative feedback reaction of thrombin generation. In order to develop optimal biological activity, a co-factor (Protein S) is necessary.
In the European patent application EP 0 406 216 a pharmaceutical preparation is described which contains Protein S, optionally, in combination with activated Protein C, and can be employed for the treatment or prevention of thrombosis and thrombo-embolic complications.
According to EP 0 519 900 the use of a Protein C-containing pharmaceutical preparation together with a thrombolytically effective substance for the treatment of thrombosis and for the prevention of re-occlusion is possible. It was found that during the thrombolysis therapy a deficiency of Protein C results wherefore the substitution with Protein C is recommended.
The effect of the inactive zymogen of Protein C differs fundamentally from the active enzyme, activated Protein C.
Activated Protein C enables the prevention of arterial thrombosis or stenosis, preferably in combination with a thrombolytically effective agent (tissue plasminogen activator, tPA); for this, see EP 0 318 201.
It is also known that in blood platelet-enriched plasma (PRP) activated Protein C suppresses platelet aggregation which is induced by thrombin activation. However, a higher concentration of activated Protein C leads to an opposite effect, namely to the aggregation of the blood platelets (E. N. Santander et al., Acta Physiologica Latino-Americana 3.3. (2), 1983).
Activated or stimulated blood platelets possess the glycoprotein IIb-IIIa complex which functions as a receptor for various adhesion molecules. Among the adhesion proteins that bind to GP IIb-IIIa of stimulated blood platelets are fibrinogen, von Willebrand Factor, and fibronectin. It is supposed that a tri-peptide sequence, namely Arg-Gly-Asp (RGD), of the adhesion proteins binds to the receptor. Among the proteins with a RGD sequence are also human Protein C and activated Protein C. However, the interaction of Protein C or activated Protein C with blood platelets is uncertain. It was found, for example, that activated Protein C binds to non-stimulated blood platelets in the presence of Protein S, and by this, the inactivation of Factor Va is potentiated. Protein C does not, however, bind to the blood platelets (J. Biol. Chem., 260 (4), 2007-10, 1985).
With the help of flow cytometry for thrombocytes, surfaces of thrombocytes can be examined. Flow Cytometry allows the fast and sensitive analysis of receptor proteins on a single cell. During the examinations, a flow rate from 400-1000 thrombocytes per second is employed. The size of the thrombocytes is expressed by the light scattering. By the use of antibodies coupled with fluorescein-isothiocyanate, the binding of ligands (adhesion proteins) on a thrombocyte can be measured (description of the method in Blood, 86 (1), 173-179, 1986).
The binding of fibrinogen to stimulated thrombocytes leads finally to the aggregation and/or deposition of these on injured endothelium and therewith to occlusion of the wound. Thrombo-embolic complications or stenosis are also attributable to the binding of fibrinogen to stimulated thrombocytes. For example, balloon angioplasty leads to an injury of the endothelium and therewith to a predisposition for arterial restenosis.